Hi All,

Below is a part of shearwater manual with example:

#-------------

3.2 More realistic example

Suppose the bam files are in folder ./bam and the regions of interest are stored in a GRanges() object with
metadata column Gene, indicating which region (typically exons for a pulldown experiment) belongs to
which gene. Also assume that we have a tabix indexed vcf file CosmicCodingMuts_v63_300113.vcf.gz.

The analysis can be parallelized by separately analysing each gene, which is the unit needed to compute
the prior using makePrior.

## Not run

files <- dir("bam", pattern = "*.bam$", full.names = TRUE)
MC_CORES <- getOption("mc.cores", 2L)
vcfList <- list()
for (gene in levels(mcols(regions)$Gene)) {
  rgn <- regions[mcols(regions)$Gene == gene]
  counts <- loadAllData(files, rgn, mc.cores = MC_CORES)

  ## Split into

  BF <- mcChunk("bbb", split = 200, counts, mc.cores = MC_CORES)
  COSMIC <- readVcf("CosmicCodingMuts_v63_300113.vcf.gz", "GRCh37", param =   
    ScanVcfParam(which = rgn))
  prior <- makePrior(COSMIC, rgn, pi.mut = 0.5)
  vcfList[[gene]] <- bf2Vcf(BF = BF, counts = counts, regions = rgn, samples = files,
    cutoff = 0.5, prior = prior)
}

## Collapse vcfList

vcf <- do.call(rbind, vcfList)

#-----------------------

In this example no information about reference genome is provided?Can anyone explains why?  is it due to bam files are already aligned files?

================

Here is another example to call variants using VariantTools where i can not see information for reference genome. Is it also due to that BAM files are aligned files??

bams <- LungCancerLines::LungCancerBamFiles()
  tally.param <- TallyVariantsParam(gmapR::TP53Genome(),
  high_base_quality = 23L,
  which = gmapR::TP53Which())

## simple usage

variants <- callVariants(bams$H1993, tally.param)

## customize

calling.filters <- VariantCallingFilters(p.error = 1/1000)
callVariants(bams$H1993, tally.param, calling.filters)

Thank you

The answer to both questions is yes. Bam files are intended to contain information about the read and where (and how well) it aligns to the reference. Any further information about the reference genome would be redundant.